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Construction and Verification of a Mouse Pulmonary Microvascular Endothelial Cell Lines to Inhabit TRPM2 Gene Expression with shRNA Lentivirus System
Liang Ting2, Li Peiyao1, Luo Qiang2 , Wang Shaohua1, Li Jun2, Xu Mingju1, Zhang Ruihua1, Xu Tong1,2*
1Key Laboratory of Preventive Veterinary Medicine, Department of Veterinary Medicine, Animal Science College, Hebei North University, Zhangjiakou 075000, China; 2Life Science Research Center, Hebei North University, Zhangjiakou 075000, China
Abstract: To construct and verify transient receptor potential melastatin-2 (TRPM2) gene silencing of mouse pulmonary microvascular endothelial cell (PMVEC)， the PMVEC was infected by short sairpin RNA (shRNA) trpm2 lentivirus particles. The results showed that the maximal tolerance concentration of TPCK-trypsin to PMVEC was 0.4 μg/mL and minimal lethal dose of puromycin dihydrochloride to PMVEC was 0.6 μg/mL， respectively. The stable clones expressing the shRNA TRPM2 were selected by adding 0.6， 2.0， 4.0， 8.0 μg/mL puromycin dihydrochloride to kill the non-transduced PMVEC， and there was no effect on PMVEC of the stable clones expressing shRNA TRPM2 even the puromycin dihydrochloride concentration reach to maximal 8 μg/mL. The result of Semi-quantitative PCR showed that the TRPM2 gene was silenced significantly in shRNA TRPM2 cell compared to shRNA control and uninfected PMVEC (P<0.01). Similarly， the protein expression of shRNA TRPM2 PMVEC was also decreased markedly compared with shRNA control and PMVEC group (P<0.01). The shRNA TRPM2 PMVEC cell line was successfully constructed by shRNA TRPM2 lentivirus particles and lie the foundation for further to investigate the mechanism of TRPM2 on PMVEC damage infected by influenza virus.
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